作物学报2012，Vol.38Issue(8)：1443-1451,9.DOI:10.3724/SP.J.1006.2012.01443
棉花非冗余性EST-SSR新标记的开发及其评价
Development and Evaluation of New Non-Redundant EST-SSR Markers from Gossypium
摘要
Abstract
A software Clustal X was used to analyse the redundancy of 393 753 ESTs of Gossypium available in public database. By mining 349 815 non-redundant ESTs, a total of 11 372 SSR loci derived from 10 507 ESTs using a software SSRmine developed by ourselves were observed. The frequency of ESTs containing SSRs was 3%, with an average of one SSR in every 21 kb of EST sequence. Besides, trinucleotide and hexanucleotide repeats were found to be the most abundant among 2-6-nucleotide repeat types, accounting for 34.1% and 40.6% respectively. In dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, pentanucleotide repeats and hexanucleotide repeats, AG/CT, AAG/CTT, AAAT/ATTT, AAAAG/CTTTT, AAAAAG/CTTTTT motifs accounted for the highest proportions, respectively. Two hundred pairs of new non-redundant EST-SSR primers were developed based on 410 EST sequences removed the redundancy which have not been developed so far in Gossypium arboreum, G. Hirsutum, and G. Barbadense. We used a software SSRmine developed by ourselves to obtain non-similarity primers, designated CRI (Cotton Research Institute) XXX through six steps, including SSR primer sequences download, pretreatment, Blastn, extraction of primer numbers of similarity score more than 81 %, extraction of redundant primers pairs and making redundant primers in a line, to remove homologous sequences from themselves and similar primers released in CMD from different cotton species. Among them, 100 primers were evaluated in polymorphism information content (PIC) and transferability using 12 cotton species including seven representative diploids species and five tetraploid species. The results showed that a total of 56 from the 100 pairs of SSR primers could be amplified the stable and clear polymorphic bands in the 12 accessions mentioned above, moreover, 35 out of 56 pairs of primers were polymorphic, with the primer polymorphism ratio of 35%. PIC of these primers ranged from 0.097 to 0.888, with the average of 0.482. Totally, the transferability among the 12 cotton species was 100% for a pair of EST-SSR primers from Gossypium barbadense L., 81% for 25 primers from G arboreum, and 80.1% for 74 primers from G hirsutum, respectively.关键词
棉花/EST-SSR标记/冗余性/多态性/通用性Key words
Gossypium/ EST-SSR marker/ Redundancy/ Polymorphism/ Transferability引用本文复制引用
王为,王长彪,刘方,陈浩东,王琳,王春英,张香娣,王玉红,王坤波..棉花非冗余性EST-SSR新标记的开发及其评价[J].作物学报,2012,38(8):1443-1451,9.基金项目
本研究由国家重点基础研究发展计划项目(973计划)(2010CB126000),中央级公益性科研院所基本科研业务专项(SJB1105),国家自然科学基金项目(30900911,31000729),江苏省科技支撑计划项目(BE2011304),江苏省农业科技自主创新基金(自由探索类),江苏沿海所所长基金项目(YHS201103)和山西省基础研究计划项目(2009021032-1). （973计划）
